Superhelical DNA as a preferential binding target of 14-3-3γ protein

Abstract

The 14-3-3 protein family is a highly conserved and widely distributed group of proteins consisting of multiple isoforms in eukaryotes. Ubiquitously expressed, 14-3-3 proteins play key roles in DNA replication, cell cycle regulation, and apoptosis. The function of 14-3-3 proteins is mediated by interaction with a large number of other proteins and with DNA. It has been demonstrated that 14-3-3γ protein binds strongly to cruciform structures and is crucial for initiating replication. In this study, we analyzed DNA binding properties of the 14-3-3γ isoform to linear and supercoiled DNA. We demonstrate that 14-3-3γ protein binds strongly to long DNA targets, as evidenced by electrophoretic mobility shift assay on agarose gels. Binding of 14-3-3γ to DNA target results in the appearance of blurry, retarded DNA bands. Competition experiments with linear and supercoiled DNA on magnetic beads show very strong preference for supercoiled DNA. We also show by confocal microscopy that 14-3-3 protein in the HCT-116 cell line is co-localized with DNA cruciforms. This implies a role for the 14-3-3γ protein in its binding to local DNA structures which are stabilized by DNA supercoiling.

Keywords:

• 14-3-3 proteins
• DNA binding
• protein-DNA complex
• superhelical DNA

Acknowledgments

This work was supported by the Grant Agency of the Czech Republic (301/10/1211), by the Ministry of Education, Youth and Sports of the Czech Republic (LC 06035), and by the Academy of Science of the Czech Republic (grant M200040904). SR was supported by a postdoctoral fellowship of the German Academic Exchange Service (DAAD).